A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2S04 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP -Sepharose. The apparent molecular weight of the reductase was found to be about 38000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1 -0.2 M citrate buffer at 30 "C. The enzyme was markedly inhibited by thiol reagents.The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest K, value (73 pM) and highest V (230 nkat/mg) followed by 5-hydroxyferuloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The K, value for NADPH varied with the type of substrate. K, values of 28, 120, and 290 pM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5 % of that found with NADPH.The reaction products CoASH and NADP' inhibited the reaction. The K, values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.

1. An 0-methyltransferase catalyzing the transfer of the S-methyl group of S-adenosyl-L-methionine to the 4'-hydroxyl group of isoflavones has been detected in seedlings of Cicer arietinum L. and in cell suspension cultures of this plant.2. After transfer of the cell cultures to fresh medium the activity of the methyltransferase rose after a lag period of about 2 h and reached a maximum after about 20 h and then declined. Illumination of the cell cultures had no effect on the enzyme activity.3. The enzyme has been purified 360-fold by (NH4)2S04 fractionation, chromatography on DEAEcellulose and hydroxyapatite and Sephadex G-200 gel filtration.4. The S-adenosylmethionine : isoflavone 4'-0-methyltransferase was isolated as a soluble enzyme with a pH optimum of 9 in glycine-NaOH buffer. It requires -SH-protecting agents for activity. The molecular weight of the enzyme was estimated by the elution volume from a Sephadex G-200 column to be about 110000.5. The enzyme is specific for the methylation of 4'-hydroxyisoflavones. Intermediates in the biosynthetic pathway to isoflavones, e.g. p-hydroxycinnamic acid and 5,7,4'-trihydroxyflavone, were not methylated. The K,,, values for daidzein (7,4'-dihydroxyisofldvone) and S-adenosylmethionine were determined to be 80 pM and 0.16 mM, respectively. Because of the high specificity of the methyltransferase for isoflavones it can be concluded that methylation is the last step in the biosynthesis of 4'-methoxyisoflavones. 6.The enzyme was inhibited by S-adenosylhomocysteine. The K i values for S-adenosylhomocysteine versus daidzein and S-adenosylmethionine were determined to be 0.3 mM and 30 pM,

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